TWI678209B - Probiotic composition for preventing, improving or alleviating pancreatic cancer and application thereof - Google Patents
Probiotic composition for preventing, improving or alleviating pancreatic cancer and application thereof Download PDFInfo
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Abstract
本發明提供一種預防、改善或減緩胰臟癌之益生菌組成物及其用途。前述益生菌組成物係由特定菌株及特定比例的副乾酪乳桿菌以及羅伊氏乳桿菌所組成，可顯著改善或減緩胰臟癌之各種病況指標及惡化程度，進而用於製備預防、改善或減緩胰臟癌的口服組成物之用途。 The present invention provides a probiotic composition for preventing, improving or slowing down pancreatic cancer and its use. The aforementioned probiotic composition is composed of a specific strain and a specific ratio of Lactobacillus paracasei and Lactobacillus reuteri, which can significantly improve or slow down various conditions and deterioration of pancreatic cancer, and is further used for preparing prevention, improvement or Use of an oral composition for slowing pancreatic cancer.
Description
本發明是有關於一種益生菌組成物及其應用，特別是有關於一種預防、改善或減緩胰臟癌之益生菌組成物及其應用。 The invention relates to a probiotic composition and its application, in particular to a probiotic composition to prevent, improve or slow down pancreatic cancer and its application.
根據台灣衛生福利部資料統計，民國105年胰臟癌為因癌症死因中的第八名。但胰臟癌是卻最易致死癌症之一，主要是因為其難以早期發現，一旦確診後，高達52%的病患已為第四期，平均病患從確診到死亡大約只有數個月的存活時間，只有少數患者透過外科手術可以獲得良好的控制。目前仍大多使用化學療法治療胰臟癌，但治療效果及五年存活率不佳，因此有必要尋找更佳的胰臟癌預防及治療之策略。 According to statistics from Taiwan ’s Ministry of Health and Welfare, pancreatic cancer was the eighth leading cause of cancer death in the Republic of China in 105 years. However, pancreatic cancer is one of the most deadly cancers, mainly because it is difficult to detect early. Once diagnosed, up to 52% of patients are in the fourth stage. The average patient has only a few months from diagnosis to death. Survival time can be well controlled by only a few patients through surgery. Currently, chemotherapy is mostly used to treat pancreatic cancer, but the treatment effect and five-year survival rate are not good. Therefore, it is necessary to find better strategies for pancreatic cancer prevention and treatment.
過去認為胰臟癌發生的危險因子可能有：過度飲酒、糖尿病、抽菸、急性及慢性胰臟炎、血型、基因遺傳 等等。根據近年的流行病學的統計調查結果，發現胰臟癌與牙周病的發生呈現正相關性。其次，在胰臟癌確診的患者血液檢體中，亦可偵測到牙周病的病原菌牙齦卟啉單胞菌(Porphyromonas gingivalis；P.gingivalis)的抗體，推測胰臟癌的惡化與此病原菌有關。 In the past, the risk factors for pancreatic cancer were thought to be: excessive drinking, diabetes, smoking, acute and chronic pancreatitis, blood type, genetic inheritance, and so on. According to the results of epidemiological statistical surveys in recent years, it was found that pancreatic cancer was positively correlated with the occurrence of periodontal disease. Secondly, antibodies to Porphyromonas gingivalis ( P. gingivalis ), a pathogen of periodontal disease, can also be detected in blood samples from patients diagnosed with pancreatic cancer. It is speculated that the deterioration of pancreatic cancer and this pathogenic bacteria related.
越來越多報導指出癌症與腸道菌相具有關聯性，而投予特定的益生菌，有機會預防及治療各式癌症。舉例來說，某些益生菌可直接殺死癌細胞。某些益生菌可經由提升宿主免疫力，以輔助免疫抑制劑之效果，另外，某些益生菌有助於預防及治療大腸直腸癌。然而，在預防及治療胰臟癌方面，與益生菌相關的實證研究則鮮少。 More and more reports indicate that cancer is related to intestinal bacteria, and the administration of specific probiotics has the opportunity to prevent and treat various types of cancer. For example, certain probiotics can directly kill cancer cells. Some probiotics can enhance the immunity of the host to assist the effect of immunosuppressive agents. In addition, some probiotics can help prevent and treat colorectal cancer. However, there are few empirical studies related to probiotics in the prevention and treatment of pancreatic cancer.
有鑑於此，實有必要提供一種益生菌組成物，以預防、改善或減緩胰臟癌的病況及惡化。 In view of this, it is necessary to provide a probiotic composition to prevent, improve or slow down the condition and worsening of pancreatic cancer.
因此，本發明之一態樣是在提供一種預防、改善或減緩胰臟癌之益生菌組成物，其包含副乾酪乳桿菌(Lactobacillus paracasei)以及羅伊氏乳桿菌(Lactobacillus reuteri)。 Therefore, one aspect of the present invention is to provide a probiotic composition for preventing, ameliorating or slowing down pancreatic cancer, which comprises Lactobacillus paracasei and Lactobacillus reuteri .
本發明之另一態樣係在提供一種益生菌組成物用於製造預防、改善或減緩胰臟癌及其併發症之組成物的用途，其係利用上述益生菌組成物以每日有效劑量向一對象經口投予達一預設期間，以降低該對象之胰臟癌的病況指標及/或指標蛋白。 Another aspect of the present invention is to provide a use of a probiotic composition for the manufacture of a composition for preventing, ameliorating or slowing down pancreatic cancer and its complications, which utilizes the above-mentioned probiotic composition at a daily effective dose to A subject is administered orally for a preset period of time to reduce the condition index and / or index protein of the subject's pancreatic cancer.
根據本發明之上述態樣，提出一種預防、改善或減緩胰臟癌之益生菌組成物，包含副乾酪乳桿菌(Lactobacillus paracasei)GMNL-133以及羅伊氏乳桿菌(Lactobacillus reuteri)GMNL-89，其中副乾酪乳桿菌GMNL-133與羅伊氏乳桿菌GMNL-89之菌數比可例如為2：1至1：2。 According to the above aspect of the present invention, a probiotic composition for preventing, ameliorating or slowing down pancreatic cancer is proposed, comprising Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89, The ratio of the bacterial counts of Lactobacillus paracasei GMNL-133 to Lactobacillus reuteri GMNL-89 may be, for example, 2: 1 to 1: 2.
在上述實施例中，副乾酪乳桿菌GMNL-133可例如為於2011年7月5日寄存於台灣新竹食品路331號財團法人食品工業發展研究所生物資源中心(BCRC)、寄存編號為BCRC910520之菌株。上述羅伊氏乳桿菌GMNL-89可例如為於2006年11月14日寄存於台灣新竹食品路331號財團法人食品工業發展研究所生物資源中心(BCRC)、寄存編號為BCRC910340之菌株。 In the above embodiment, Lactobacillus paracasei GMNL-133 may be deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on July 5, 2011, and the deposit number is BCRC910520. Strains. The aforementioned Lactobacillus reuteri GMNL-89 may be, for example, a strain deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan, on November 14, 2006, and the deposit number is BCRC910340.
在本發明的一實施例中，上述副乾酪乳桿菌GMNL-133與羅伊氏乳桿菌GMNL-89之菌數比可例如為1：1。 In an embodiment of the present invention, the ratio of the bacterial count of the Lactobacillus paracasei GMNL-133 to the Lactobacillus reuteri GMNL-89 may be, for example, 1: 1.
根據本發明之另一態樣，提出一種益生菌組成物用於製造預防、改善或減緩胰臟癌及其併發症之組成物的用途，包含向一對象經口投予每日有效劑量8.2×108個菌落形成單位(colony forming unit；CFU)/kg體重之上述益生菌組成物達4週，以降低該對象之胰臟癌的病況指標及/或指標蛋白。 According to another aspect of the present invention, a use of a probiotic composition for manufacturing a composition for preventing, ameliorating or slowing pancreatic cancer and its complications is provided, which comprises orally administering to a subject an effective daily dose of 8.2 × 108 colony forming units (colony forming unit; CFU) / kg body weight of the probiotic composition described above for 4 weeks to reduce pancreatic cancer condition index of the subject and / or metrics protein.
在本發明的一實施例中，上述組成物為口服組成物。 In one embodiment of the present invention, the composition is an oral composition.
應用本發明之預防、改善或減緩胰臟癌之益生菌組成物，其中所含特定菌株的副乾酪乳桿菌以及羅伊氏乳桿菌可顯著改善胰臟癌之各種病況指標及惡化程度，進而應用於預防、改善或減緩療胰臟癌的口服組成物。 By applying the probiotic composition for preventing, improving or slowing pancreatic cancer, the specific strains of Lactobacillus paracasei and Lactobacillus reuteri can significantly improve various disease indexes and exacerbations of pancreatic cancer. Oral composition for preventing, improving or slowing down pancreatic cancer.
為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之詳細說明如下：〔圖1〕係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟重量散布圖。 In order to make the above and other objects, features, advantages, and embodiments of the present invention more comprehensible, the detailed description of the drawings is as follows: [FIG. 1] Shows pancreatic cancer mice according to an embodiment of the present invention. Scatter diagram of pancreatic weight after administration of the probiotic composition.
〔圖2A〕至〔圖2C〕係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟組織免疫染色切片圖。 [FIG. 2A] to [FIG. 2C] are diagrams showing pancreas tissue immunostained sections after administration of a probiotic composition to a pancreatic cancer mouse according to an embodiment of the present invention.
〔圖3A〕至〔圖3C〕係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟增殖細胞核抗原(proliferating cell nuclear antigen；PCNA)的組織免疫染色切片圖。 [FIG. 3A] to [FIG. 3C] Tissue immunostained sections showing pancreatic proliferating cell nuclear antigen (PCNA) after administration of a probiotic composition to pancreatic cancer mice according to an embodiment of the present invention Illustration.
〔圖4A〕至〔圖4F〕係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟膠原蛋白的組織免疫染色切片圖。 [Fig. 4A] to [Fig. 4F] are immunohistochemical sections of pancreas collagen after administration of a probiotic composition to pancreatic cancer mice according to an embodiment of the present invention.
〔圖5A〕至〔圖5L〕係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟N-Cad(圖5A至圖5C)、中間絲蛋白(vimentin；圖5D至圖5F)、Snail-1(圖5G至圖5I)及ZEB(圖5J至圖5L)的組織免疫染色切片圖。 [Fig. 5A] to [Fig. 5L] shows pancreas N-Cad (Fig. 5A to Fig. 5C) and intermediate filament protein (vimentin) after administration of a probiotic composition to a pancreatic cancer mouse according to an embodiment of the present invention Figures 5D to 5F), Snail-1 (Figures 5G to 5I), and ZEB (Figures 5J to 5L) tissue immunostaining sections.
〔圖6A〕至〔圖6F〕係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物的胰臟TGF-β(圖6A至圖6C)及活化態(磷酸化)Smad3(phospho-Smad3；圖6D至圖6F)蛋白的組織免疫染色切片圖。 [Fig. 6A] to [Fig. 6F] shows pancreatic TGF-β (Fig. 6A to Fig. 6C) and activated state (phosphorylation) of a pancreatic cancer mouse according to an embodiment of the present invention after administration of a probiotic composition Tissue immunostaining sections of Smad3 (phospho-Smad3; Figure 6D to Figure 6F) proteins.
本發明所提到的單數形式“一”、“一個”和“所述”包括複數引用，除非上下文另有明確規定。數值範圍(如10%~11%的A)若無特定說明皆包含上、下限值(即10%≦A≦11%)；數值範圍若未界定下限值(如低於0.2%的B，或0.2%以下的B)，則皆指其下限值可能為0(即0%≦B≦0.2%)。上述用語是用以說明及理解本發明，而非用以限制本發明。 The singular forms "a", "an" and "the" mentioned in the present invention include plural references unless the context clearly indicates otherwise. The numerical range (such as 10% ~ 11% A) includes the upper and lower limit values (ie, 10% ≦ A ≦ 11%) if there is no specific description; if the numerical range does not define a lower limit value (such as B below 0.2%) , Or B) below 0.2%, it means that its lower limit may be 0 (that is, 0% ≦ B ≦ 0.2%). The above terms are used to explain and understand the present invention, but not to limit the present invention.
本發明提供一種預防、改善或減緩胰臟癌之益生菌組成物，其包含特定菌株的副乾酪乳桿菌以及羅伊氏乳桿菌，可顯著預防、改善或減緩胰臟癌之各種病況指標及惡化程度。 The invention provides a probiotic composition for preventing, improving or slowing down pancreatic cancer, which comprises a specific strain of Lactobacillus paracasei and Lactobacillus reuteri, which can significantly prevent, improve or slow down various disease indexes and deterioration of pancreatic cancer. degree.
在一實施例中，上述特定菌株的副乾酪乳桿菌以及羅伊氏乳桿菌係指副乾酪乳桿菌(Lactobacillus paracasei)GMNL-133以及羅伊氏乳桿菌(Lactobacillus reuteri)GMNL-89。具體而言，上述副乾酪乳桿菌GMNL-133係指於2011年7月5日寄存於台灣新竹食品路331號財團法人食品工業發展研究所生物資源中心(BCRC)、寄存編號為BCRC910520之菌株。上述羅伊 氏乳桿菌GMNL-89係指於2006年11月14日寄存於台灣新竹食品路331號財團法人食品工業發展研究所生物資源中心(BCRC)、寄存編號為BCRC910340之菌株。 In one embodiment, the specific strains of Lactobacillus paracasei and Lactobacillus reuteri refer to Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89. Specifically, the above-mentioned Lactobacillus paracasei GMNL-133 refers to the strain deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on July 5, 2011, and the deposit number is BCRC910520. The above-mentioned Lactobacillus reuteri GMNL-89 refers to the strain deposited at the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331 Food Road, Hsinchu, Taiwan on November 14, 2006, and the deposit number is BCRC910340.
在上述實施例中，益生菌組成物的副乾酪乳桿菌GMNL-133與羅伊氏乳桿菌GMNL-89之菌數比可例如為2：1至1：2。在一例示中，副乾酪乳桿菌GMNL-133與羅伊氏乳桿菌GMNL-89之菌數比可例如為1：1。 In the above embodiment, the ratio of the bacterial count of Lactobacillus paracasei GMNL-133 to Lactobacillus reuteri GMNL-89 in the probiotic composition may be, for example, 2: 1 to 1: 2. In one example, the bacterial count ratio of Lactobacillus paracasei GMNL-133 to Lactobacillus reuteri GMNL-89 may be, for example, 1: 1.
在本發明的一實施例中，上述益生菌組成物可有效預防、改善或減緩胰臟癌的病況指標及其惡化程度。 In an embodiment of the present invention, the above-mentioned probiotic composition can effectively prevent, improve or slow down the condition index of pancreatic cancer and its deterioration degree.
本發明此處所述之病況指標可包括但不限於抑制胰臟腫大、減緩胰臟癌小鼠之腫瘤形成(例如胰腺上皮內瘤、腺癌)、抑制胰臟細胞增生、降低胰臟纖維化(例如降低膠原蛋白的表現量)、抑制癌症轉移指標蛋白(例如N-Cad、Vimentin、Snail-1及ZEB)的表現量。 The conditions described herein can include, but are not limited to, inhibition of pancreatic enlargement, slowing tumor formation in pancreatic cancer mice (e.g., pancreatic intraepithelial neoplasia, adenocarcinoma), inhibition of pancreatic cell proliferation, and reduction of pancreatic fibers (For example, reducing the expression of collagen) and suppressing the expression of cancer metastasis index proteins (for example, N-Cad, Vimentin, Snail-1, and ZEB).
本發明此處所述之惡化程度可包括但不限於透過調節免疫路徑(例如促進TGF-β的表現量及活化其下游的活化態(磷酸化)Smad3蛋白之表現量)，以改善或減緩胰臟癌惡化程度。 The degree of deterioration described herein in the present invention may include, but is not limited to, improving or slowing the pancreas by regulating the immune pathway (such as promoting the expression of TGF-β and activating the expression of the activated (phosphorylated) Smad3 protein downstream of it). Exacerbation of dirty cancer.
在一實施例中，上述益生菌組成物可進一步用於製造組成物，以預防、改善或減緩胰臟癌及/或併發症。在上述實施例中，前述組成物為口服組成物。在一例示中，上述併發症可包含但不限於胰腺上皮內瘤、腺癌、胰臟纖維化及胰臟癌相關之免疫病症等。 In one embodiment, the above-mentioned probiotic composition can be further used for manufacturing a composition to prevent, improve or slow down pancreatic cancer and / or complications. In the above embodiment, the aforementioned composition is an oral composition. In one example, the above complications may include, but are not limited to, pancreatic intraepithelial neoplasia, adenocarcinoma, pancreatic fibrosis, and pancreatic cancer-related immune disorders.
在本發明的一實施例中，上述組成物可包括但 不限於上述混合比例之副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89，並添加醫藥學上可接受的載劑。當醫藥組成物為內用組成物時，則該載劑可為以水為主的載劑，例如生理緩衝液，並經由口服方式投予。 In an embodiment of the present invention, the above composition may include but It is not limited to the above-mentioned mixing ratio of Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89, and a pharmaceutically acceptable carrier is added. When the pharmaceutical composition is a composition for internal use, the carrier may be a water-based carrier, such as a physiological buffer solution, and is administered orally.
在本發明的一實施例中，上述益生菌組成物用於製造預防、改善或減緩胰臟癌及其併發症之組成物時，上述益生菌組成物之菌體濃度並無特別限制，端視受試者的體重而調整，其每日有效劑量可例如8.2×108個菌落形成單位(colony forming unit；CFU)/kg體重。以小鼠(平均體重例如20g)為例，益生菌組成物之總菌量可例如為0.5×107菌落形成單位(colony forming unit；CFU)至3.0×107CFU為宜，又以1.64×107CFU為較佳，然不限於此。 In an embodiment of the present invention, when the above-mentioned probiotic composition is used to manufacture a composition for preventing, improving or slowing pancreatic cancer and its complications, the bacterial cell concentration of the above-mentioned probiotic composition is not particularly limited. The weight of the subject is adjusted, and the effective daily dose may be, for example, 8.2 × 10 8 colony forming unit (CFU) / kg body weight. Taking mice (average body weight, for example, 20 g) as an example, the total amount of the probiotic composition may be, for example, 0.5 × 10 7 colony forming unit (CFU) to 3.0 × 10 7 CFU, and 1.64 × 10 7 CFU is better, but it is not limited to this.
以下利用數個實施例以說明本發明之應用，然其並非用以限定本發明，本發明技術領域中具有通常知識者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾。 The following uses several embodiments to illustrate the application of the present invention, but it is not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. Retouch.
以下實施例係評估副乾酪乳桿菌(Lactobacillus paracasei；GMNL-133)及羅伊氏乳桿菌(Lactobacillus reuteri；GMNL-89)之菌學特徵。 The following examples evaluate the mycological characteristics of Lactobacillus paracasei (GMNL-133) and Lactobacillus reuteri (GMNL-89).
副乾酪乳桿菌GMNL-133之來源為人類小腸腸道。 The source of Lactobacillus paracasei GMNL-133 is the human small intestine.
副乾酪乳桿菌GMNL-133的外觀特徵如下。副乾酪乳桿菌GMNL-133接種於(De Man,Rogosa and Sharpe；MRS)瓊脂培養基上，於37℃培養48小時後，觀察其形態。菌落外觀、大小及顏色：邊緣完整，平均大小為2.5mm x 2.5mm，牛奶白色，表面平滑***。格蘭氏染色：陽性。型態：桿狀(桿菌；bacilli)。孢子形成：無。移動性：無。 The appearance characteristics of Lactobacillus paracasei GMNL-133 are as follows. Lactobacillus paracasei GMNL-133 was inoculated on (De Man, Rogosa and Sharpe; MRS) agar medium, and after culturing at 37 ° C for 48 hours, the morphology was observed. Colony appearance, size and color: the edges are complete, the average size is 2.5mm x 2.5mm, the milk is white, and the surface is smooth and raised. Gram stain: positive. Type: rod-shaped (bacilli; bacilli). Spore formation: None. Mobility: None.
副乾酪乳桿菌GMNL-133的生理特徵如下。生長溫度：35℃至40℃。生長酸鹼值：pH 4.0至7.0。氧氣影響：兼性厭氧(facultative anaerobic)。 The physiological characteristics of Lactobacillus paracasei GMNL-133 are as follows. Growth temperature: 35 ° C to 40 ° C. Growth pH: pH 4.0 to 7.0. Oxygen effect: facultative anaerobic.
另外，利用習知方法萃取副乾酪乳桿菌GMNL-133的總RNA後，藉由如序列辨識編號(SEQ ID NO)：1及2的引子對(上游引子為PAF primer，下游引子為536R primer)擴增16S rRNA基因的部分序列，其中所得之核酸片段如序列辨識編號(SEQ ID NO)：3所示。關於萃取總RNA的方法誠屬本發明所屬技術領域具有通常知識者所熟知，在此不另贅述。 In addition, after the total RNA of Lactobacillus paracasei GMNL-133 was extracted by a conventional method, primer pairs such as sequence identification numbers (SEQ ID NO): 1 and 2 were used (the upstream primer was PAF primer, and the downstream primer was 536R primer). A partial sequence of the 16S rRNA gene was amplified, and the obtained nucleic acid fragment is shown as a sequence identification number (SEQ ID NO): 3. The method for extracting total RNA is well known to those having ordinary knowledge in the technical field to which the present invention pertains, and will not be repeated here.
此核酸片段與美國國家生物技術資訊中心(National Center for Biotechnology Information；NCBI)基因庫(GenBank)之二種副乾酪乳桿菌16S rRNA基因序列(GenBank編號：NR_041054.1，NR_025880.1)比對具有100%的序列一致性(identity)。另外，序列辨識 編號(SEQ ID NO)：3與另二種副乾酪乳桿菌16S rRNA基因序列(GenBank編號：NR_113337.1，NR_117987.1)比對後，也具有99%的序列一致性，確認為副乾酪乳桿菌。 This nucleic acid fragment is aligned with two types of Lactobacillus paracasei 16S rRNA gene sequences (GenBank number: NR_041054.1, NR_025880.1) of the National Center for Biotechnology Information (NCBI) gene bank (GenBank). 100% sequence identity. In addition, sequence recognition (SEQ ID NO): 3 also has 99% sequence identity after alignment with the other two Lactobacillus paracasei 16S rRNA gene sequences (GenBank numbers: NR_113337.1, NR_117987.1), which is confirmed as Paracasein milk Bacillus.
羅伊氏乳桿菌(Lactobacillus reuteri；GMNL-89)之來源為人類小腸腸道。 Lactobacillus reuteri (GMNL-89) is derived from the human small intestine.
羅伊氏乳桿菌GMNL-89的外觀特徵如下。羅伊氏乳桿菌GMNL-89接種於MRS洋菜培養基上，於37℃培養48小時後，觀察其形態。菌落外觀、大小及顏色：邊緣完整，平均大小為2.5mm x 2.5mm，牛奶白色，表面平滑***。格蘭氏染色：陽性。型態：桿狀(桿菌)。孢子形成：無。移動性：無。 The appearance characteristics of Lactobacillus reuteri GMNL-89 are as follows. Lactobacillus reuteri GMNL-89 was inoculated on MRS agar culture medium, and after culturing at 37 ° C for 48 hours, the morphology was observed. Colony appearance, size and color: the edges are complete, the average size is 2.5mm x 2.5mm, the milk is white, and the surface is smooth and raised. Gram stain: positive. Type: rod-shaped (bacilli). Spore formation: None. Mobility: None.
羅伊氏乳桿菌GMNL-89的生理特徵如下。生長溫度：35℃至40℃。生長酸鹼值：pH 4.0至7.0。氧氣影響：兼性厭氧。 The physiological characteristics of Lactobacillus reuteri GMNL-89 are as follows. Growth temperature: 35 ° C to 40 ° C. Growth pH: pH 4.0 to 7.0. Oxygen effect: Facultative anaerobic.
另外，利用習知方法萃取羅伊氏乳桿菌GMNL-89的總RNA後，藉由如SEQ ID NO：1及2的引子對擴增16S rRNA基因的部分序列，其中所得之核酸片段如序列辨識編號(SEQ ID NO)：4所示。關於萃取總RNA的方法誠屬本發明所屬技術領域具有通常知識者所熟知，在此不另贅述。 In addition, after the total RNA of Lactobacillus reuteri GMNL-89 was extracted by a conventional method, a partial sequence of the 16S rRNA gene was amplified by primer pairs such as SEQ ID NOs: 1 and 2, and the obtained nucleic acid fragments such as sequence recognition No. (SEQ ID NO): 4 shown. The method for extracting total RNA is well known to those having ordinary knowledge in the technical field to which the present invention pertains, and will not be repeated here.
此核酸片段與NCBI基因庫之二種羅伊氏乳桿菌16S rRNA基因序列(GenBank編號：NR_075036.1， NR_113820.1，NR_119069.1，其中NR_075036.1與NR_119069.1為同一種菌株)比對後，具有99%的序列一致性，確認為羅伊氏乳桿菌。 This nucleic acid fragment and two kinds of 16S rRNA gene sequences of Lactobacillus reuteri in NCBI gene bank (GenBank No .: NR_075036.1, NR_113820.1, NR_119069.1, of which NR_075036.1 and NR_119069.1 are the same strain). After alignment, they have 99% sequence identity and are confirmed as Lactobacillus reuteri.
此實施例係利用LSL-Kras G12D 基因轉殖小鼠(以下簡稱K基因型小鼠)及LSL-Kras G12D ；Pdx-1-Cre基因轉殖小鼠(以下簡稱KC基因型小鼠)，評估益生菌組成物預防、改善或減緩胰臟癌的病況指標及調節TGF-β相關訊息傳遞路徑的效果。 This example uses LSL-Kras G12D transgenic mice (hereinafter referred to as K genotype mice) and LSL-Kras G12D ; Pdx-1-Cre transgenic mice (hereinafter referred to as KC genotype mice) to evaluate Probiotic composition prevents, improves or slows down the disease index of pancreatic cancer and regulates the effect of TGF-β-related signaling pathways.
上述K基因型小鼠與KC基因型小鼠(雄雌各半)係購自突變鼠資源及研究中心〔Mutant Mouse Resource & Research Centers，MMRRC；網址：https：//www.mmrrc.org/；舊稱為美國國家癌症中心(National Cancer Institute；NCI)小鼠儲存庫(mouse repository)〕之品系B6.129-Kras tm4Tyj (即具有LSL-Kras G12D 基因；K基因型小鼠)與B6.FVB-Tg(Ipf1-cre)1Tuv(即具有Pdx-1-Cre基因；C基因型小鼠)，經基因背景淨化為B6基因型背景，並將此二基因型小鼠進行雜交，以PCR方式進行基因型鑑定，挑選帶有LSL-Kras G12D 基因轉殖小鼠(K基因型小鼠)及LSL-Kras G12D 及Pdx-1-Cre雙基因轉殖小鼠(即具有LSL-Kras G12D 及Pdx-1-Cre基因雙基因；KC基因型小鼠)進行實驗，並依循國立成功大學及中山醫學大學實驗動物照 護及使用委員會訂立的要點及台灣動物保護法的相關規定進行。 The above-mentioned K genotype mice and KC genotype mice (male and female) were purchased from Mutant Mouse Resource & Research Centers (MMRRC; Website: https://www.mmrrc.org/; Formerly known as the National Cancer Institute (NCI) mouse repository, the strain B6.129- Kras tm4Tyj (that is, the mouse with the LSL-Kras G12D gene; K genotype) and B6.FVB -Tg (Ipf1-cre) 1Tuv (that is, Pdx-1-Cre gene; C genotype mouse), purified by genetic background to B6 genotype background, and hybridize the two genotype mice by PCR genotyping, selection and LSL-Kras G12D and Pdx-1-Cre transgenic mice with bis LSL-Kras G12D transgenic mice (K genotype mice) (i.e., having a LSL-Kras G12D and Pdx- 1-Cre gene double gene; KC genotype mouse) to conduct experiments, and follow the points set by the Experimental Animal Care and Use Committee of National Cheng Kung University and Sun Yat-Sen Medical University and the relevant provisions of the Taiwan Animal Protection Law.
簡言之，上述基因轉殖小鼠係由Cre基因控制轉殖基因表達與否。K基因型小鼠帶有LSL-Kras G12D 基因，但因無表達Cre基因，所以Kras G12D 不會大量表現，可作為無胰臟癌之控制組小鼠。 In short, the above-mentioned transgenic mice are controlled by the Cre gene for the expression of the transgenic genes. K genotype mice carry the LSL-Kras G12D gene, but because they do not express the Cre gene, Kras G12D does not express a large amount, and can be used as a control group of mice without pancreatic cancer.
KC基因型小鼠之細胞具有LSL-Kras G12D ；Pdx-1-Cre基因，可於胰臟細胞大量表達Kras G12D 蛋白質，於高齡KC基因型小鼠可自發性產生胰臟癌病變。KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌(Porphyromonas gingivalis)後，可加速促進誘發胰臟癌惡化，故作為胰臟癌之實驗組(或稱處理組)小鼠。 Cells of KC genotype mice have LSL-Kras G12D ; Pdx-1-Cre gene, which can express Kras G12D protein in pancreatic cells in large numbers, and in elderly KC genotype mice can spontaneously produce pancreatic cancer lesions. KC genotype mice can accelerate the induction of pancreatic cancer by applying Porphyromonas gingivalis to the mouth, so they are used as the experimental group (or treatment group) of pancreatic cancer mice.
進行實驗前，上述小鼠先自由飲用含抗生素之飲水10天，每5天更換一次。第11天更換為無菌逆滲透(R.O.)水休息3日以排除殘留的抗生素。處理組的小鼠牙齦及口腔黏膜塗抹牙齦卟啉單胞菌(P.gingivalis；1×109CFU/小鼠)，每2天塗抹(感染)一次，共感染三次，以加速誘導胰臟癌發生。接著，每天經口投予(即管餵)由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89組成之益生菌組合物(總菌量為1.64×107CFU/小鼠，或8.2×108CFU/kg體重，二菌株的菌數比為1：1)。4週後犧牲小鼠。 Before the experiment, the mice were free to drink antibiotic-containing water for 10 days, and replaced every 5 days. Day 11 was replaced with sterile reverse osmosis (RO) water for 3 days to exclude residual antibiotics. The mice in the treatment group were smeared with P. gingivalis ( P.gingivalis ; 1 × 10 9 CFU / mouse) on the gums and oral mucosa of the mice, and smeared (infected) every 2 days for three infections to accelerate the induction of pancreatic cancer occur. Next, a probiotic composition consisting of Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89 was administered orally (i.e., tube-fed) daily (total bacteria volume was 1.64 × 10 7 CFU / mouse, or 8.2 × 10 8 CFU / kg body weight, the ratio of the two strains was 1: 1). Mice were sacrificed after 4 weeks.
取小鼠胰臟秤重後，將胰臟組織固定、包埋、切片，進行蘇木紫及伊紅(Hematoxylin & Eosin)染色、馬森三色(Masson’s trichrome)染色(主要觀察膠原蛋白) 以及特定蛋白〔例如增殖細胞核抗原(proliferating cell nuclear antigen；PCNA)、N-鈣黏蛋白(N-cadherin；N-Cad)、中間絲蛋白(vimentin)、轉錄因子Snail-1、鋅指E-盒結合同源框(zinc finger E-box binding homeobox；ZEB)蛋白、TGF-β、磷酸化Smad3(phospho-Smad3)蛋白〕的組織免疫染色，並由專業人員判定實驗數據。 After the pancreas of the mouse was weighed, the pancreatic tissue was fixed, embedded, and sectioned, and hematoxylin & Eosin staining and Masson ’s trichrome staining were performed (mainly to observe collagen). And specific proteins such as proliferating cell nuclear antigen (PCNA), N-cadherin (N-Cad), vimentin, transcription factor Snail-1, zinc finger E-box Tissue immunostaining combined with zinc finger E-box binding homeobox (ZEB) protein, TGF-β, and phosphorylated Smad3 (phospho-Smad3) protein, and experimental data will be determined by professionals.
請參閱圖1，其係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟重量散布圖。在圖1中，縱軸代表胰臟重量(g)，橫軸則代表各處理組，其中圖號+代表經該項處理，圖號-代表未經該項處理，圖號○代表未處理之K基因型小鼠的胰臟重量，圖號●代表未處理之KC基因型小鼠的胰臟重量，圖號■代表以牙齦卟啉單胞菌處理之K基因型小鼠的胰臟重量，圖號▲代表以牙齦卟啉單胞菌處理之KC基因型小鼠的胰臟重量，圖號▼代表以牙齦卟啉單胞菌處理後投予益生菌組成物之K基因型小鼠的胰臟重量，而圖號◆代表以牙齦卟啉單胞菌處理後投予益生菌組成物之KC基因型小鼠的胰臟重量，圖號「＊」代表該二組平均值±標準差彼此之間具有統計上的顯著性差異(P<0.05)，圖號「＊＊」代表該二組平均值±標準差彼此之 間具有統計上的顯著性差異(P<0.01)，圖號「＊＊＊＊」代表該二組平均值±標準差彼此之間具有統計上的顯著性差異(P<0.0001)，ns(not significant)代表該二組平均值±標準差彼此之間不具有統計上的顯著性差異(P>0.05)，n代表之該組的樣本數。 Please refer to FIG. 1, which shows a scattergram of pancreas weight of a pancreatic cancer mouse according to an embodiment of the present invention after administration of a probiotic composition. In Figure 1, the vertical axis represents pancreatic weight (g), and the horizontal axis represents each treatment group, where the figure number + represents the treatment, and the figure number-represents the absence of this treatment, and the figure number ○ represents the untreated one. Pancreas weight of K genotype mice, drawing number ● represents pancreatic weight of untreated KC genotype mice, drawing number ■ represents pancreatic weight of K genotype mice treated with P. gingivalis, Figure number ▲ represents pancreas weight of KC genotype mice treated with P. gingivalis, figure number ▼ represents pancreas of K genotype mice administered with probiotic composition after treatment with P. gingivalis Dirty weight, and the figure number ◆ represents the pancreas weight of the KC genotyped mice administered with the probiotic composition after treatment with P. gingivalis, and the figure number "*" represents the mean and standard deviation of the two groups. There is a statistically significant difference between them ( P <0.05), and the plot number "**" represents the statistical significance of the two groups of mean ± standard deviation ( P <0.01), and the plot number "**" *** "represents the statistically significant difference between the mean and standard deviation of the two groups ( P <0.0001), ns (not s ignificant) represents the mean ± standard deviation of the two groups without statistically significant difference ( P > 0.05), and n represents the number of samples in the group.
由圖1之圖號○及圖號●的結果顯示，未處理之KC基因型小鼠的胰臟重量增加程度，大於未處理之K基因型小鼠的胰臟重量，顯示Kras G12D 大量表現會使得胰臟重量增加。 Figure number is shown by ○ and ● in the drawing number of the results of FIG. 1, the untreated weight of the pancreas to increase the degree of KC genotype mice, the pancreas is greater than the weight of the untreated mice genotypes K, will exhibit large display Kras G12D Makes pancreas weight increase.
其次，由圖1之圖號●及圖號▲的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌誘發胰臟癌後，使得胰臟重量增加或腫大更為顯著，顯示牙齦卟啉單胞菌的確能加重胰臟癌的惡性程度。 Secondly, the results of Figure No. ● and Figure No. ▲ in Fig. 1 show that after KC genotypes were applied to the oral cavity to induce pancreatic cancer by Porphyromonas gingivalis, the weight or swelling of the pancreas became more significant. Porphyromonas gingivalis can indeed aggravate the malignancy of pancreatic cancer.
然而，由圖1之圖號▲及圖號◆的結果顯示，口服益生菌組合物能夠降低KC基因型小鼠塗抹牙齦卟啉單胞菌所造成的胰臟重量增加或腫大，平均值甚至略低於未處理組的範圍值(圖號●與圖號◆的結果)，雖然以胰臟重量來說不具有統計意義(例如圖號●與圖號◆的結果彼此之間為ns，不具有統計上的顯著性差異)，證明由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89組成之益生菌組合物確實能抑制小鼠胰臟重量增加或腫大。 However, the results of Figure No. ▲ and Figure No. ◆ in Figure 1 show that the oral probiotic composition can reduce the weight or swelling of the pancreas caused by Porphyromonas gingivalis in KC genotype mice. The average value is even Slightly lower than the range value of the untreated group (result of plot number ● and plot number ◆), although it is not statistically significant in terms of pancreatic weight (for example, the results of plot number ● and plot number ◆ are ns between each other, not There is a statistically significant difference), proving that a probiotic composition consisting of Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89 can indeed inhibit the increase or swelling of mouse pancreas weight.
請參閱圖2A至圖2C，其係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟組織免疫染色切片圖，其中圖2A代表未處理之KC基因型小鼠的胰臟組織免疫染色切片圖，圖2B代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後的胰臟組織免疫染色切片圖，圖2C代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物的胰臟組織免疫染色切片圖，BPC代表良性胰臟細胞(benign pancreatic cells)，PanIns代表胰腺上皮內瘤(Pancreatic Intraepithelial Neoplasia)，AC代表腺癌細胞(adenocarcinoma cells)。 Please refer to FIG. 2A to FIG. 2C, which are pancreas tissue immunostaining sections after administration of a probiotic composition to a pancreatic cancer mouse according to an embodiment of the present invention, where FIG. 2A represents an untreated KC genotype Immunostaining section of pancreas tissue in mice. Fig. 2B represents an immunostaining section of pancreas tissue after KC genotypes were smeared with Porphyromonas gingivalis in the mouth. Immunostaining section of pancreas tissue after administration of Porphyromonas to the probiotic composition, BPC stands for benign pancreatic cells, PanIns stands for Pancreatic Intraepithelial Neoplasia, and AC stands for adenocarcinoma (adenocarcinoma cells).
由圖2A及圖2B的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌誘發胰臟癌後，胰臟組織出現較大量癌化區塊，如圖2B所示。 The results of FIG. 2A and FIG. 2B show that after KC genotypes were applied to the oral cavity to induce pancreatic cancer induced by Porphyromonas gingivalis, a large number of cancerous blocks appeared in the pancreatic tissue, as shown in FIG. 2B.
然而，由圖2C的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物，則無腺癌細胞(AC)生成，證明由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89組成之益生菌組合物確實可明顯改善或減緩胰臟癌之腫瘤形成。 However, the results shown in FIG. 2C show that when KC genotype mice were administered a probiotic composition after oral application of Porphyromonas gingivalis, no adenocarcinoma cells (AC) were produced, which proved that Lactobacillus paracasei GMNL-133 And the probiotic composition composed of Lactobacillus reuteri GMNL-89 can obviously improve or slow the tumor formation of pancreatic cancer.
請參閱圖3A至圖3C，其係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟增殖細胞核抗原(proliferating cell nuclear antigen；PCNA)的組織免疫染色切片圖，其中圖3A代表未處理之KC基因型 小鼠的胰臟PCNA組織免疫染色切片圖，圖3B代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後的胰臟PCNA組織免疫染色切片圖，圖3C代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物的胰臟PCNA組織免疫染色切片圖。 Please refer to FIG. 3A to FIG. 3C, which are tissue immunostaining sections of pancreatic proliferating cell nuclear antigen (PCNA) after administration of a probiotic composition to pancreatic cancer mice according to an embodiment of the present invention. Figure, where Figure 3A represents untreated KC genotype Immunostaining section of pancreas PCNA tissue in mice, Fig. 3B represents the immunostaining section of pancreas PCNA tissue after KC genotype mice were smeared with Porphyromonas gingivalis in the mouth, and Fig. 3C represents KC genotype mice in the mouth Immunostaining section of pancreas PCNA tissue after administration of a probiotic composition after smearing P. gingivalis.
由圖3A至圖3B的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌誘發胰臟癌後，胰臟組織出現較大量PCNA，代表胰臟細胞大量增生，如圖3B所示。 The results from Figs. 3A to 3B show that after KC genotypes applied P. gingivalis to the pancreas cancer induced by pancreas cancer, a large amount of PCNA appeared in the pancreatic tissue, which represented a large number of pancreatic cell proliferation, as shown in Fig. 3B .
然而，由圖3C的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物，則胰臟的增生指標PCNA的表現量明顯降低，證明由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89組成之益生菌組合物確實可改善或減緩胰臟癌小鼠胰臟之細胞增生。 However, according to the results of FIG. 3C, when the KC genotype was administered with a probiotic composition after oral application of Porphyromonas gingivalis to the oral cavity, the expression of the pancreatic proliferation index PCNA was significantly reduced. The probiotic composition composed of GMNL-133 and Lactobacillus reuteri GMNL-89 can indeed improve or slow down the pancreatic cell proliferation of pancreatic cancer mice.
請參閱圖4A至圖4F，其係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟膠原蛋白的組織免疫染色切片圖，其中圖4A至4C依序對應於圖4D至圖4F，惟圖4A至圖4C之比例尺為600μm，而圖4D至圖4F之比例尺為150μm。圖4A及圖4D代表未處理之KC基因型小鼠的胰臟膠原蛋白組織免疫染色切片圖，圖4B及圖4E代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後的胰臟膠原蛋白組織免疫染色切片圖，圖4C及圖4F代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物的胰臟 膠原蛋白組織免疫染色切片圖。 Please refer to FIG. 4A to FIG. 4F, which are immunohistochemical sections of pancreas collagen after administration of a probiotic composition to pancreatic cancer mice according to an embodiment of the present invention, in which FIGS. 4A to 4C are sequentially Corresponding to FIGS. 4D to 4F, the scales of FIGS. 4A to 4C are 600 μm, and the scales of FIGS. 4D to 4F are 150 μm. Figures 4A and 4D represent pancreas collagen tissue immunostaining sections of untreated KC genotype mice, and Figures 4B and 4E represent pancreas collagen of KC genotype mice after oral application of P. gingivalis Protein tissue immunostaining sections, Figures 4C and 4F represent pancreas of KC genotype mice administered with probiotic composition after oral application of P. gingivalis Collagen tissue immunostaining section.
由圖4A、圖4B、圖4D及圖4E的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌誘發胰臟癌後，胰臟組織出現較大量膠原蛋白，代表胰臟纖維化，如圖4B及圖4E所示。 From the results of FIGS. 4A, 4B, 4D and 4E, after KC genotypes were applied to the oral cavity of Porphyromonas gingivalis to induce pancreatic cancer, a larger amount of collagen appeared in pancreatic tissues, representing pancreatic fibrosis. , As shown in Figure 4B and Figure 4E.
然而，由圖4C及圖4F的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物，則胰臟的膠原蛋白之表現量明顯降低，證明由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89組成之益生菌組合物確實可改善或減緩胰臟癌小鼠的胰臟纖維化之惡化程度。 However, the results of FIGS. 4C and 4F show that when the KC genotype is administered with a probiotic composition after oral application of Porphyromonas gingivalis, the expression of collagen in the pancreas is significantly reduced. The probiotic composition composed of Lactobacillus GMNL-133 and Lactobacillus reuteri GMNL-89 can indeed improve or slow down the degree of pancreatic fibrosis in pancreatic cancer mice.
請參閱圖5A至圖5L，其係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物後的胰臟N-Cad(圖5A至圖5C)、中間絲蛋白(vimentin；圖5D至圖5F)、Snail-1(圖5G至圖5I)及ZEB(圖5J至圖5L)的組織免疫染色切片圖。圖5A、圖5D、圖5G及圖5J代表未處理之KC基因型小鼠的胰臟組織免疫染色切片圖，圖5B、圖5E、圖5H及圖5K代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後的胰臟組織免疫染色切片圖，圖5C、圖5F、圖5I及圖5L代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物的胰臟組織免疫染色切片圖。 Please refer to FIG. 5A to FIG. 5L, which show pancreatic N-Cad (FIG. 5A to FIG. 5C), intermediate filament protein (vimentin) after pancreatic cancer mice are administered with a probiotic composition according to an embodiment of the present invention. Figures 5D to 5F), Snail-1 (Figures 5G to 5I), and ZEB (Figures 5J to 5L) tissue immunostaining sections. Figures 5A, 5D, 5G, and 5J represent pancreas tissue immunostaining sections of untreated KC genotype mice, and Figures 5B, 5E, 5H, and 5K represent KC genotype mice smearing gums in the mouth Immunostaining section of pancreas tissue after Porphyromonas. Figures 5C, 5F, 5I and 5L represent pancreas administered to the probiotic composition by the KC genotype mice after Porphyromonas gingivalis was applied to the oral cavity. Immunostaining section of dirty tissue.
由圖5A、圖5B、圖5D及圖5E的結果顯示，KC 基因型小鼠於口腔塗抹牙齦卟啉單胞菌誘發胰臟癌後，胰臟組織出現大量表現的N-Cad及中間絲蛋白等間葉組織指標(mesnchymal markers)，如圖5B及圖5E所示，代表胰臟癌細胞已轉型成較易轉移之型態。其次，由圖5G、圖5H、圖5J及圖5K的結果顯示，胰臟組織出現大量表現的Snail-1及ZEB等促癌轉移蛋白，如圖5H及圖5K所示，代表胰臟癌細胞已轉型成較惡性且較易轉移之型態。 5A, 5B, 5D and 5E show that KC After genotyping mice with Porphyromonas gingivalis to induce pancreatic cancer, a large number of mesenchymal markers such as N-Cad and intermediate filament proteins appeared in pancreatic tissues, as shown in Figures 5B and 5E. It shows that pancreatic cancer cells have transformed into a more metastatic form. Secondly, the results of Figures 5G, 5H, 5J, and 5K show that pancreatic tissues have a large number of tumor-promoting proteins such as Snail-1 and ZEB, as shown in Figures 5H and 5K, which represent pancreatic cancer cells It has been transformed into a more vicious and easier to transfer form.
然而，由圖5C、圖5F、圖5I及圖5L的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物，則胰臟的N-Cad、中間絲蛋白、Snail-1及ZEB的表現量明顯降低，證明由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89組成之益生菌組合物確實可改善或減緩胰臟癌化之惡性程度。 However, the results of FIGS. 5C, 5F, 5I, and 5L show that when the KC genotype is administered with a probiotic composition after Porphyromonas gingivalis is administered to the oral cavity, the pancreatic N-Cad and intermediate filaments The expression levels of protein, Snail-1 and ZEB were significantly reduced, proving that a probiotic composition composed of Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89 could indeed improve or slow down the malignant degree of pancreatic cancer.
在胰臟癌的進程中，調控免疫系統的細胞激素也扮演相當重要的角色，其中TGF-β及其上下游的訊號傳遞路徑有可能是促使胰臟癌惡化的關鍵。 In the process of pancreatic cancer, cytokines that regulate the immune system also play a very important role. Among them, TGF-β and its upstream and downstream signal transmission pathways may be the key to promoting pancreatic cancer.
請參閱圖6A至圖6F，其係顯示根據本發明一實施例之胰臟癌小鼠經投予益生菌組成物的胰臟轉化生長因子(transforming growth factor；TGF)-β(圖6A至圖6C)及磷酸化Smad3(phospho-Smad3；圖6D至圖6F)蛋白的組織免疫染色切片圖，圖6A及圖6D代表未處理之KC基因 型小鼠，圖6B及圖6E代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後的胰臟組織免疫染色切片圖，圖6C及圖6F代表KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物的胰臟組織免疫染色切片圖。 Please refer to FIG. 6A to FIG. 6F, which show pancreatic transforming growth factor (TGF) -β after administration of a probiotic composition to pancreatic cancer mice according to an embodiment of the present invention. 6C) and phosphorylated Smad3 (phospho-Smad3; Figure 6D to Figure 6F) protein immunostaining sections, Figures 6A and 6D represent untreated KC gene 6B and FIG. 6E represent pancreas tissue immunostaining sections of KC genotype mice after oral application of P. gingivalis, and FIGS. 6C and 6F represent KC genotype mice after oral application of gingival porphyries. Immunostaining sections of pancreatic tissues after administration of the probiotic composition to morphomonas.
由圖6A、圖6B、圖6D及圖6E的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌誘發胰臟癌後，胰臟組織出現大量表現的TGF-β(如圖6B所示)並活化(磷酸化)其下游的Smad3蛋白(如圖6E所示)。 From the results of Figs. 6A, 6B, 6D, and 6E, after pancreatic cancer induced by Porphyromonas gingivalis in KC genotype mice, a large number of TGF-β appeared in pancreatic tissues (Fig. 6B) (Shown) and activates (phosphorylates) its downstream Smad3 protein (shown in Figure 6E).
然而，由圖6C及圖6F的結果顯示，KC基因型小鼠於口腔塗抹牙齦卟啉單胞菌後投予益生菌組合物，則胰臟組織的TGF-β(如圖6C所示)及其下游的磷酸化Smad3蛋白(如圖6F所示)的表現量明顯減少，證明由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89組成之益生菌組合物可能經由TGF-β相關訊息傳遞路徑改善或減緩胰臟癌惡化之惡性程度。 However, according to the results of FIG. 6C and FIG. 6F, when the KC genotype is administered with a probiotic composition after oral application of Porphyromonas gingivalis, the TGF-β of the pancreatic tissue (as shown in FIG. 6C) and The expression level of its downstream phosphorylated Smad3 protein (shown in Figure 6F) was significantly reduced, proving that a probiotic composition composed of Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89 may be related via TGF-β The message pathway improves or slows the malignancy of pancreatic cancer.
由上述結果顯示，本發明以LSL-Kras G12D 基因轉殖鼠作為動物模式，利用牙齦卟啉單胞菌(P.gingivalis)誘發胰臟癌後，的確產生各種胰臟癌的相關症狀(其病況指標可例如胰臟重量增加、胰臟組織癌化及過度增生)以及胰臟癌惡性程度加遽(其惡性程度可例如胰臟纖維化程度增加、胰臟癌症轉移指標增加)。上述胰臟過度纖維化會相對抑制抗癌藥物之治療效果，而胰臟癌轉移則是胰臟癌高惡性程度之主因。然而，給予本發明之益生菌組成物，其由副乾酪乳桿菌GMNL-133及/或羅伊氏乳桿菌GMNL-89所組 成，可顯著改善或減緩上述胰臟癌之病況指標及其惡化程度。 From the above results, it is shown that the present invention uses LSL-Kras G12D gene transgenic mice as an animal model, and after using P. gingivalis to induce pancreatic cancer, it does produce various pancreatic cancer-related symptoms (its condition Indicators can be, for example, increased pancreatic weight, pancreatic tissue canceration and hyperplasia), and increased malignant degree of pancreatic cancer (the malignant degree can be, for example, increased degree of pancreatic fibrosis, increased index of pancreatic cancer metastasis). The above-mentioned excessive fibrosis of the pancreas will relatively inhibit the therapeutic effect of anticancer drugs, and pancreatic cancer metastasis is the main cause of the high malignancy of pancreatic cancer. However, the probiotic composition provided by the present invention, which is composed of Lactobacillus paracasei GMNL-133 and / or Lactobacillus reuteri GMNL-89, can significantly improve or slow down the above-mentioned indicators of pancreatic cancer conditions and the extent of their deterioration. .
本發明也證實，由副乾酪乳桿菌GMNL-133及羅伊氏乳桿菌GMNL-89所組成之益生菌組成物可經由調節胰臟之TGF-β表現量，抑制其下游磷酸化Smad蛋白的訊息傳遞，進而達到免疫調節之功效，甚至改善胰臟癌的進程及惡化。其次，副乾酪乳桿菌GMNL-133及/或羅伊氏乳桿菌GMNL-89源於人體腸道，相當安全且不具有副作用，確實有潛力應用於預防及治療胰臟癌的口服組成物。 The invention also confirms that the probiotic composition composed of Lactobacillus paracasei GMNL-133 and Lactobacillus reuteri GMNL-89 can inhibit the message of phosphorylated Smad protein downstream by regulating the expression of TGF- β in the pancreas Transmission, and then achieve the effect of immune regulation, and even improve the progress and worsening of pancreatic cancer. Secondly, Lactobacillus paracasei GMNL-133 and / or Lactobacillus reuteri GMNL-89 are derived from the human intestinal tract, are quite safe and have no side effects, and indeed have potential applications in oral compositions for the prevention and treatment of pancreatic cancer.
補充說明的是，雖有研究結果顯示，有些乳酸菌可藉由抑制牙齦卟啉單胞菌(P.gingivalis)之生長而抑制牙周病，但根據本發明前期試驗結果顯示，本發明之益生菌組成物對於牙齦卟啉單胞菌(P.gingivalis)並無明顯抑菌能力(圖未顯示)，因此排除本發明之益生菌組成物經由直接抑制牙齦卟啉單胞菌(P.gingivalis)之生長而達到改善胰臟癌病況的可能性。 It is added that although some research results show that some lactic acid bacteria can inhibit periodontal disease by inhibiting the growth of P. gingivalis , according to the preliminary test results of the present invention, the probiotics of the present invention show that The composition does not have significant antibacterial activity against P. gingivalis (not shown in the figure), so the probiotic composition of the present invention is excluded from directly inhibiting P. gingivalis by The possibility of growing to improve the condition of pancreatic cancer.
綜言之，本發明雖以特定菌株組合、特定的比例、特定的投予方式、或特定的評估方式作為例示，說明本發明之預防、改善或減緩胰臟癌之益生菌組成物及其用途，惟本發明所屬技術領域中任何具有通常知識者可知，本發明並不限於此，在不脫離本發明之精神和範圍內，本發明亦可使用其他菌株組合、其他的比例、其他的投予方式或其他的評估方式進行。 In summary, although the present invention exemplifies a specific strain combination, a specific ratio, a specific administration method, or a specific evaluation method, the probiotic composition of the present invention for preventing, improving or slowing pancreatic cancer and its use However, anyone with ordinary knowledge in the technical field to which the present invention belongs can know that the present invention is not limited to this, and the present invention can also use other combinations of strains, other ratios, and other administrations without departing from the spirit and scope of the present invention. Or other assessment methods.
由上述實施例可知，本發明之預防、改善或減 緩胰臟癌之益生菌組成物及其用途，其優點在於此益生菌組成物係由特定菌株及特定比例的副乾酪乳桿菌以及羅伊氏乳桿菌所組成，可顯著改善或減緩胰臟癌之各種病況指標及惡化程度，進而用於製備預防、改善或減緩胰臟癌的口服組成物之用途。 It can be known from the above embodiments that the prevention, improvement or reduction of the present invention Probiotic composition for slowing pancreatic cancer and its use. The advantage is that this probiotic composition is composed of a specific strain and a specific proportion of Lactobacillus paracasei and Lactobacillus reuteri, which can significantly improve or slow down pancreatic cancer. The various indicators of the condition and the degree of deterioration are further used for preparing an oral composition for preventing, improving or slowing down pancreatic cancer.
雖然本發明已以數個實施例揭露如上，然其並非用以限定本發明，在本發明所屬技術領域中任何具有通常知識者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。 Although the present invention has been disclosed as above with several embodiments, it is not intended to limit the present invention. Any person with ordinary knowledge in the technical field to which the present invention pertains can make various modifications without departing from the spirit and scope of the present invention. Changes and retouching, so the protection scope of the present invention shall be determined by the scope of the appended patent application.
副乾酪乳桿菌GMNL-133寄存於中華民國財團法人食品工業發展研究所生物資源保存及研究中心，寄存日期為2011年7月5日，寄存編號為BCRC910520。 Lactobacillus paracasei GMNL-133 was deposited at the Center for Bioresources Preservation and Research of the Food Industry Development Institute of the Republic of China. The deposit date is July 5, 2011, and the deposit number is BCRC910520.
羅伊氏乳桿菌GMNL-89寄存於中華民國財團法人食品工業發展研究所生物資源保存及研究中心，寄存日期為2006年11月14日，寄存編號為BCRC910340。 Lactobacillus reuteri GMNL-89 was deposited at the Biological Resource Preservation and Research Center of the Food Industry Development Institute of the Republic of China. The deposit date is November 14, 2006, and the deposit number is BCRC910340.
<110> 景岳生物科技股份有限公司 <110> Jingyue Biological Technology Co., Ltd.
<120> 預防、改善或減緩胰臟癌之益生菌組成物及其應用 <120> Probiotic composition for preventing, improving or slowing pancreatic cancer and application thereof
<130> 無 <130> None
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<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
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<223> 副乾酪乳桿菌(Lactobacillus paracasei)16S rDNA部分序列 <223> Lactobacillus paracasei 16S rDNA partial sequence
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<400> 4
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| TWI346554B (en) * | 2008-04-30 | 2011-08-11 | Genmont Biotech Inc | Lactobacillus isolates having anti-inflammatory activities and uses of the same |
| TWI398259B (en) * | 2011-10-14 | 2013-06-11 | Genmont Biotech Inc | Composition and use of lactobacillus paracasei strain gmnl-133 in treating atopic dermatitis or other allergic diseases |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI346554B (en) * | 2008-04-30 | 2011-08-11 | Genmont Biotech Inc | Lactobacillus isolates having anti-inflammatory activities and uses of the same |
| TWI398259B (en) * | 2011-10-14 | 2013-06-11 | Genmont Biotech Inc | Composition and use of lactobacillus paracasei strain gmnl-133 in treating atopic dermatitis or other allergic diseases |
Non-Patent Citations (4)
| Title |
|---|
| JEREMY A. PEÑA, "Probiotic Lactobacillus spp. Diminish Helicobacter hepaticus-Induced Inflammatory Bowel Disease in Interleukin-10-Deficient Mice", INFECTION AND IMMUNITY, Vol.73, No.2, Feb. 2005, pages 912-920. * |
| MING-SHIOU JAN, "Probiotics ameliorate Porphyromonas gingivalis-promoted pancreatic cancer progression in oncogenic Kras transgenic mice [abstract]". In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5 * |
| MING-SHIOU JAN, "Probiotics ameliorate Porphyromonas gingivalis-promoted pancreatic cancer progression in oncogenic Kras transgenic mice [abstract]". In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 235. |
| Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 235. * |
Also Published As
| Publication number | Publication date |
|---|---|
| TW201940183A (en) | 2019-10-16 |
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